Two Distinct Human POM121 Genes: Requirement for the Formation of Nuclear Pore Complexes

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 581 (2007) 4910–4916 Two distinct human POM121 genes: Requirement for the formation of nuclear pore complexes Tomoko Funakoshia, Kazuhiro Maeshimaa, Kazuhide Yahatab, Sumio Suganoc, Fumio Imamotob, Naoko Imamotoa,* a Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan b Department of Molecular Biology, BIKEN, Osaka University, Osaka, Japan c The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Received 30 March 2007; revised 12 September 2007; accepted 12 September 2007 Available online 21 September 2007 Edited by Ulrike Kutay essential in somatic cells, as this protein is not ubiquitously ex- Abstract Pom121 is one of the integral membrane components of the nuclear pore complex (NPC) in vertebrate cells. Unlike ro- pressed in mammalian cells [6]. Pom121 is recruited at an early dent cells carrying a single POM121 gene, human cells possess stage of post-mitotic nuclear reassembly in vertebrate cells, multiple POM121 gene loci on chromosome 7q11.23, as a conse- and its turnover on the interphase NPC is very slow [7]. These quence of complex segmental-duplications in this region during findings make Pom121 a strong candidate that could possess human evolution. In HeLa cells, two ‘‘full-length’’ Pom121 are essential roles in anchoring the NPC to the NE. Indeed, transcribed and translated by two distinct genetic loci. RNAi Pom121 was shown to be essential for NPC formation, but experiments showed that efficient depletion of both Pom121 pro- not gp210 in the Xenopus egg extracts [8]. The human ortholog teins significantly reduces assembled NPCs on nuclear envelope. of yeast NDC1, which was initially isolated as a component of Pom121-depletion also induced clustering of NPCs, indicating spindle pole bodies in yeast, localizes to the NE in HeLa cells its role on maintenance of NPC structure/organization. during interphase [9,10]. Recently, two groups reported the Ó 2007 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. essential roles of Ndc1 for NPC assembly in mammalian cells, but controversial results were reported for Pom121 [8,9,11].It Keywords: Nuclear pore complex; Pom121; Nuclear structure; is still unclear whether Pom121 is necessary for NPC assembly RNAi; Segmental-duplication or not in mammalian somatic cells. In this study, we found that human ‘‘full-length’’ Pom121 are encoded by two distinct genetic loci, both of which are transcribed and translated in HeLa cells. Using several RNAi oligos targeted to both or either of Pom121 transcripts, we 1. Introduction show that depletion of Pom121 causes a reduction of assembled NPCs, and clustering of NPCs. Our results show necessity Nuclear pore complex (NPC) mediates the transport of mac- of Pom121 on NPC assembly and maintenance of NPC struc- romolecules across the nuclear envelope (NE) [1]. The NPC is ture/organization. a very large protein complex with an estimated mass of 60– 125 MDa in vertebrates and is assembled from multiple copies of approximately 30 different proteins termed nucleoporins 2. Materials and methods (Nups) [2]. NPCs are dynamic structures that undergo assembly and disassembly upon cell cycle progression. NPC assem- 2.1. Isolation of two distinct human POM121 cDNAs and expressions of bly takes place not only at the end of mitosis when the NE their Venus-fusion genes in HeLa cells reforms around daughter chromosomes, but also during inter- POM121 cDNA was obtained from HeLa S3 total RNA using the 50 phase on the assembled NE [3,4]. RACE system (Invitrogen) according to the manufacture’s instructions. First-strand cDNA was synthesized with SuperScriptä III re- The integral membrane Nups are believed to play an impor- verse transcriptase (Invitrogen) and POM121 specific primer tant role in NPC formation and anchoring of the other Nups (Supplementary materials). Poly-dA tail was added to the first-strand to the NE. In vertebrates, only three Nups, Pom121, gp210 cDNA and used as a template for second-strand synthesis using an oli- and NDC1 are known as transmembrane proteins. gp210 is go-(dT)17 primer. POM121 cDNAs were amplified by PCR with LA- Taq DNA polymerase (TaKaRa) using primer sets corresponding to implicated in NPC formation, since an antibody against its POM1211–24 and POM121837–855 at locus A, and POM1211–24 and short C-terminal peptide and the peptide itself both inhibited POM1211180–1201 at locus C. The amplified DNA-fragments were ver- NPC formation in the Xenopus egg extract [5]. However, the ified by DNA sequencing and fused with a truncated POM121 cDNA function of gp210 in NPC assembly is considered to be non- clone (GenBank accession nos: BC008794) to obtain cDNA coding the full-length of human Pom121 proteins. For the expression of POM121 cDNAs, pEXPR-PEF-1a-POM121A-Venus, and pEXPR-PEF-a- *Corresponding author. Fax: +81 48 462 4715. POM121NC-Venus were constructed based on multi-site Gateway E-mail address: [email protected] (N. Imamoto). system (Invitrogen) as described previously [3,12]. To express POM121-Venus and CFP simultaneously, tandem expression clones, Abbreviations: NPC, nuclear pore complex; NE, nuclear envelope; pEXPR-PEF-1a-POM121A-Venus-Ix2-PEF-1a-SECFP, pEXPR-PEF-1a- Nup, nucleoporin; RT-PCR, reverse transcriptase-polymerase chain POM121NC-Venus-Ix2-PEF-1a-SECFP were constructed with tandem reaction; siRNA, small interfering RNA insulator sequences (Ix2) that ensure comparable expressions of 0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.09.021 T. Funakoshi et al. / FEBS Letters 581 (2007) 4910–4916 4911 POM121-Venus and SECFP [13]. Ix2 insulator consists of two repeti- the C-terminal portion of rodent Pom121, but its predicted N- tive chicken b-globin HS4 insulators (a gift from Dr. Felsenfeld). Sta- terminal sequence shows no homology with Pom121. This is ble and transient transformants expressing POM121-Venus were due to a truncation of segmental-duplication block at the cen- obtained as previously described [3,12]. tromeric terminal on locus B. A deduced 1229-aa polypeptide 2.2. RNAi transfection encoded by locus C harbors an N-terminal hydrophobic trans- The cells plated at 1 · 104 cells in 24-well plates were transfected membrane domain, but no information of a cDNA encoding with 50 nM small interfering RNAs (siRNAs) with Oligofectamineä ‘‘full-length’’ Pom121 protein derived from this locus is avail- reagent (Invitrogen) according to the manufacture’s instructions. After able in the databases with the exception of deduced sequences 72 h, the cells were processed for indirect immunofluorescence. For from the genome. studies in Fig. 3, cells were synchronized by double thymidine treatment (2 mM, 16 h) in order to obtain uniform expression levels and nuclear rim targeting of transfected Pom121. The cells on a 35 mm-dish 3.2. Identification and expression analysis of a new exon were co-transfected with 2 lg plasmid and 48 nM RNA oligo, after encoding a transmembrane domain of POM121 1st thymidine treatments. Twenty-seven hours after 2nd thymidine The lack of the 50-portion of human POM121 cDNA that treatment (44-h after co-transfection), middle section images (0.2 lm) were obtained. Fluorescence intensities of Venus of nuclear rims of encodes the N-terminal region containing the transmembrane the cells expressing SECFP at almost the same level were measured domain prompted us to re-examine the genetic loci. We sur- using SoftWorx software package (Applied Precision). veyed the genomic sequence of locus A that shows homology The sequences of siRNAs used are indicated in Supplementary mate- with the N-terminal region of rodent Pom121. A candidate se- rials. quence was present within a long intron between the original exon 4 and exon 5 (Fig. 1B). The sequence segment encodes 2.3. Immunofluorescence staining Immunofluorescence was carried out as described previously [3] ex- an ORF homologous to the first 189 amino acids of rodent cept that methanol-fixed cells were used in all cases. Antibody dilution Pom121 protein including the transmembrane region (supple- rates used are following: 1:3000 dilution for mAb414 (Covance, MMS- mentary Fig. S1C). Exon/intron boundary consensus se- 120P); 1:1000 dilution for Pom121, lamin A/C (Santa Cruz, sc-20681), quences were found on both sides of this segment and the and goat secondary antibodies conjugated with Alexa Fluor 488, 594, and 647 (Invitrogen). spliced junction sequence followed the GT-AG rule, indicating that this region contains an exon that was dismissed in the pre- vious annotation. We referred this putative exon as exon 4a. A 3. Results and discussion genomic sequence similar to exon 4a also exists in locus C, but not in locus B (supplementary Fig. S1B). Successful amplifica- 3.1. Human POM121 is assigned on three genetic loci on tion of three small regions using reverse transcriptase-polymer- chromosome 7q11.23 ase chain reaction (RT-PCR) from locus A and C, which Pom121 is a well-conserved transmembrane NPC compo- contain small ‘‘gaps’’ missing in one gene from another, indi- nent present in a wide range of vertebrate species, and is cated that ‘‘full-length’’ POM121 from both locus A and C thought to serve as the anchoring sites of the vertebrate are transcribed in HeLa cells (supplementary Fig. S2A, B). NPC to the lipid bilayers. To obtain a putative complete se- In addition, several unique peptide fragments corresponding quence of the human POM121 gene, we searched the Gen- to the both POM121 products were detected by mass spec- Bank/EMBL databases. Unlike rodent cells carrying a single trometry analysis of nuclear pore fraction isolated from HeLa POM121 gene, we found that human cells possess three differ- cells (supplementary Fig.

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